Systems and methods for quantifying an analyte extracted from a sample

ABSTRACT

The invention generally relates to systems and methods for quantifying an analyte extracted from a sample. In certain embodiments, the invention provides methods that involve introducing a solvent into a capillary, introducing the capillary into a vessel including a sample such that a portion of the sample is introduced into the capillary, moving the sample and the solvent within the capillary to induce circulation within the sample and the solvent, thereby causing the analyte to be extracted from the sample and into the solvent, analyzing the analyte that has been extracted from the sample, and quantifying the analyte. In certain embodiments, the quantifying step is performed without knowledge of a volume of the sample and/or solvent.

RELATED APPLICATION

The present application is a continuation of U.S. nonprovisionalapplication Ser. No. 16/293,017, filed Mar. 5, 2019, which is acontinuation of U.S. nonprovisional application Ser. No. 15/064,865,filed Mar. 9, 2016, which is a continuation-in-part of PCT/US15/13649,filed Jan. 30, 2015, which claims the benefit of and priority to U.S.provisional patent application Ser. No. 61/942,949, filed Feb. 21, 2014,and U.S. provisional patent application Ser. No. 62/013,007, filed Jun.17, 2014. The present application also claims the benefit of andpriority to U.S. provisional application Ser. No. 62/130,024, filed Mar.9, 2015. The content of each of these applications is incorporated byreference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under GM106016 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The invention generally relates to systems and methods for quantifyingan analyte extracted from a sample.

BACKGROUND

Rapid analysis of complex biological samples (e.g., complex mixturessuch as blood, saliva, or urine) is of significant interest forclinical, forensic and many other applications. A problem in theanalysis of such samples using mass spectrometry is that non-targetcomponents of a biological sample (e.g., salts), compete with a targetin the sample for charges during the ionization process. Thatcompetition leads to the non-target components of the sample suppressingionization of the target in the sample, known as matrix effects. Inorder to minimize suppression effects on analyte ionization and topre-concentrate the analytes, complex biological samples are routinelyextracted and then separated using chromatography before a massspectrometry measurement. However, such a process can only be conductedin a laboratory setting using expensive chromatography equipment andtime consuming sample preparation protocols.

SUMMARY

The invention provides a new approach for liquid-liquid extractions andalso provides systems and methods that allow for sample preparation andpre-treatment to be combined with the ionization process. Particularly,the invention allows for a liquid-liquid extraction to be conducted in acapillary of very small diameter, which provides a very small interfacebetween the two liquids (e.g., capillary of inner diameter as small as500 um). That is typically considered highly unfavorable for traditionalliquid-liquid extractions. The liquid in the capillary is moved back andforth, which allows for control of the extraction process, i.e., theextraction can be turned on and off. The motion induces circulationinside each plug of immiscible fluid, which facilitates the extraction.The added benefits for using the thin capillary are that small amountsof samples (e.g., large volume samples of greater than 100 μl) can behandled for analysis and further for quantitative analysis.

Additionally, it is possible for the extraction capillary to serve as anionization probe. In that manner, the invention provides systems andmethods that allow a target analyte in a sample to be extracted andanalyzed by mass spectrometry without conducting separate samplepreparation and pre-treatment protocols. Rather, systems and methods ofthe invention are configured so that sample preparation andpre-concentration are conducted within the ionization probe. Thepurified analyte can then be directly ionized (although not required)and injected into a mass spectrometer from the ionization probe in whichthe sample preparation and pre-treatment occurred.

In certain embodiments, aspects of the invention are accomplished usinga solvent that is immiscible with the sample. A solvent is introducedinto a capillary. The capillary is introduced into a vessel including asample such that a portion of the sample is introduced into thecapillary. The sample and the solvent are moved within the capillary toinduce circulation within the sample and the solvent, thereby causingthe analyte to be extracted from the sample and into the solvent. Themethods of the invention can be performed with any volume of sample.Methods of the invention may additionally involve quantifying theanalyte. In certain embodiments, the quantifying step is performedwithout knowledge of a volume of the sample. In certain embodiments, thesample is a large volume sample (e.g., a sample having a volume greaterthan 100 μl).

In embodiments in which the extraction capillary also serves as theionization probe, an electrode is then operably coupled to the solventwithin the body of the ionization probe (capillary) and the targetanalyte is ionized and injected into the mass spectrometer. In thatmanner, sample preparation and pre-treatment are combined with theionization process, and when voltage is applied to the solvent, thetarget analyte does not need to compete with the non-target componentsof the sample (e.g., salts in urine) for charges. According to suchembodiments, systems and methods of the invention effectively suppressmatrix effects, and allow for better ionization of target analytes fromsamples, particularly biological samples, such as blood, saliva, urine,or spinal cord fluid. Systems and methods of the invention also have theadded benefit of pre-concentrating target analytes from a sample intothe extraction solvent, thereby avoiding expensive chromatographyequipment and time consuming separation protocols, and enablingpoint-of-care sample analysis systems and methods.

One of skill in the art will recognize that the order in which thesample and the solvent are introduced to the hollow body (e.g.,capillary) does not matter. In certain embodiments, the solvent isintroduced first and the sample is introduced second. In otherembodiments, the sample is introduced first and the solvent isintroduced second. In certain embodiments, the sample and the solventare immiscible. In certain embodiments, more than one solvent is used.

For example, a second solvent can be introduced that sits between thefirst solvent and the sample (three phase embodiments). The secondsolvent can act as a solvent bridge, and is immiscible with the sampleand the first solvent, which are typically miscible with each other insuch an embodiment.

In certain embodiments, the sample and the solvent are gently mixedprior to application of the voltage. That can be conducted manually, bygently tilting the capillary, or through use of a moving mechanism. Incertain embodiments, the two or more phases do not mix with each other.An exemplary moving mechanism is a pump that applies altering pneumaticforces within the hollow body to push and pull the sample within thebody, thereby causing gentle movements. In certain embodiments, a highvoltage was applied to the solvent eject the solvent from the hollowbody, desolvate and ionize the sample. In other embodiments, anebulizing gas is also applied to the extracted sample, either pulsed oras a continuous flow.

Methods of the invention can be used with any type of sample. In certainembodiments, the sample is a biological fluid, such as blood, urine,saliva, or spinal cord fluid. The sample will typically include a targetof interest. In the case of biological samples, that target may be atherapeutic drug, a drug of abuse, or other molecule, such as a steroid.The target may be a component that is native to the sample, or one thathas been artificially introduced to the sample. In certain embodiments,an internal standard is also introduced.

In certain embodiments, the target to be analyzed is a target that isnot efficiently ionized, for example, by spray ionization. In suchembodiments, it is beneficial to derivatize the target molecule byintroduce an agent that is able to impart a charged group to the target,thereby making it more amenable to ionization. For example, steroids aredifficult to ionize by spray ionization. Introducing an agent to thesample, such as hydroxylamine, imparts a charged group to the steroid,making it amenable to spray ionization.

The extraction solvent chosen will depend on the target to be extracted.It is important to also consider the effectiveness of the solvent forionization. An ideal solvent is good for both extraction and ionization.Such an exemplary solvent is ethyl acetate, although the skilled artisanwill recognize that other solvents are also effective to both extractand ionize a target in a sample. In embodiments in which multipleanalytes are extracted from the sample into the solvent, the solvent mayalso be able to differentially extract the analytes.

Numerous methods exist for analyzing the ions. In certain embodiments,analyzing involves introducing the ions to a mass analyzer of a massspectrometer or a miniature mass spectrometer. An exemplary miniaturemass spectrometer is described, for example in Gao et al. (Anal. Chem.2008, 80, 7198-7205), the content of which is incorporated by referenceherein in its entirety. In comparison with the pumping system used forlab-scale instruments with thousands watts of power, miniature massspectrometers generally have smaller pumping systems, such as a 18 Wpumping system with only a 5 L/min (0.3 m3/hr) diaphragm pump and a 11L/s turbo pump for the system described in Gao et al. Other exemplaryminiature mass spectrometers are described for example in Gao et al.(Anal. Chem.,2006, 80:7198-7205, 2008), Hou et al. (Anal. Chem.,83:1857-1861, 2011), and Sokol et al. (Int. J. Mass Spectrom., 2011,306, 187-195), the content of each of which is incorporated herein byreference in its entirety.

Other aspects of the invention provide systems for analyzing an analytein a sample. The system includes an ionization probe and a massspectrometer. As mentioned above, the mass spectrometer may be abench-top mass spectrometer or a miniature mass spectrometer. Theionization probe includes a hollow body that includes a distal tip(e.g., a glass capillary stretched to a tip). The hollow body isconfigured such that there is no substrate within the body and noelectrode disposed on a surface of the body. Rather, an electrode is atleast partially disposed within the hollow body. In certain embodiments,the electrode is spaced from the surfaces of the hollow body, i.e., doesnot touch the surfaces of the hollow body. In certain embodiments, theelectrode is coaxially disposed within the hollow body. In certainembodiments, the electrode extends to a distal portion of the hollowbody. An exemplary electrode is a metal wire.

In certain embodiments, the probe operates without pneumatic assistance.In other embodiments, the system includes a source of nebulizing gas.The source of nebulizing gas may be configured to provide pulses of gasor may be configured to provide a continuous flow of gas.

In other aspects, the invention provides methods for extracting ananalyte from a sample. Those methods involve introducing a solvent intoa capillary. A sample including an analyte is also introduced into thecapillary. In certain embodiments, the solvent does not mix with thesample. In other embodiments, the solvent and the sample to mix witheach other. The sample and the solvent are moved within the capillary toinduce circulation within the sample and the solvent, thereby causingthe analyte to be extracted from the sample and into the solvent. Incertain embodiments, the solvent is introduced first and the sample isintroduced second. In other embodiments, the sample is introduced firstand the solvent is introduced second. In certain embodiments, the sampleand the solvent are immiscible. In certain embodiments, more than onesolvent is used. For example, a second solvent can be introduced thatsits between the first solvent and the sample (three phase embodimentsusing a bridging solvent). The second solvent can act as a solventbridge, and is immiscible with the sample and the first solvent, whichare typically miscible with each other in such an embodiment.

In certain embodiments, the methods may additionally involve analyzingthe extracted analyte. Analyzing may involve applying a voltage to thesolvent comprising the extracted analyte in the capillary so that theanalyte is expelled from the capillary, thereby generating ions of theanalyte, and analyzing the ions. In other embodiments, analyzing mayinvolve removing the solvent comprising the extracted analyte from thecapillary, and conducting an assay that analyzes the analyte.

Methods of the invention allow for the reactions to be monitored. Tomonitor the reaction, the methods may additionally involve stoppingmovement of the sample and the solvent within the capillary, andanalyzing an amount of analyte that has been extracted into the solvent.Based on the results of the analyzing step, the methods of the inventionmay additionally involve re-starting movement of the sample and thesolvent within the capillary based on results of the analyzing step.

Another aspect of the invention provides methods for extracting ananalyte from a sample that involve introducing multiple solvents into acapillary, in which each two adjacent solvents do not mix with eachother, and moving the solvents within the capillary to inducecirculation within each solvent, thereby causing the chemical compoundsto be transferred between the solvents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an exemplary system of the invention.

FIG. 1B shows a method of using systems of the invention. In thisfigure, two immiscible phases, the liquid sample and the organic solventare injected adjacently in a capillary with a pulled tip. The liquidphases are moved back and forth in the capillary, by tilting thecapillary or applying gas pressure, to facilitate the microextraction.The liquid phases are then pushed with the extraction phase reaching thepulled tip of the capillary, by applying a gas pressure, and a wireelectrode is inserted into the extraction solvent to apply a DC voltagefor nanoESI.

FIG. 2 panels A-C show calibration curves for quantitation of differentcompounds in synthetic urine samples. FIG. 2 panel A showsmethamphetamine. FIG. 2 panel B shows nicotine. FIG. 2 panel C showsbenzoylecgonine. 10 μL synthetic urine containing the drugs and internalstandards were used as samples for the measurement. 5 μL ethyl acetate(EA) was used as the extraction phase for extraction, purification andspray. Internal standards: methamphetamine-d8 at 0.8 ng/mL, nicotine-d32at ng/mL, benzoylecgonine-d3 at 1 ng/mL. The single reaction monitoring(SRM) transitions used: methamphetamine m/z 150→91, methamphetamine-d8m/z 158→93; nicotine 163→130, nicotine-d3 m/z 166→130; benzoylecgoninem/z 290→168, benzoylecgonine-d3 m/z 293→171. Partition coefficients:LogPmethamphetamine=2.07; LogPnicotine=1.17, LogPbenzoylecgonine=-0.59.

FIG. 3A shows reactive slug flow microextraction nanoESI with a samplephase, a derivatization reagent phase and an extraction phase injectedadjacently. The extraction phase is immiscible with the reagent phase orthe sample phase; the sample phase and the reagent phase can bemiscible. The analytes in the sample phase are derivatized and extractedinto the extraction phase during the SFME operation. The liquid phasesare then pushed so the extraction phase reaches the pulled tip of thecapillary and a wire electrode is inserted into the extraction phase toapply the DC spray voltage for nanoESI.

FIG. 3B shows the binding of a charged reagent to a target in a sampleto form a charged complex, which is more amenable to spray ionizationthan the uncharged target.

FIG. 4 panels A-B show MS/MS spectra obtained using slug flowmicroextraction nanoESI. Bovine blood samples, each containing 40 ng/mLnicotine (FIG. 4 panel A) and 40 ng/mL methamphetamine (FIG. 4 panel B)were diluted 10 times with water and then analyzed using SFME nanoESI.10 μL of diluted sample, 5 μL ethyl acetate used.

FIG. 5 panels A-D show MS/MS spectra obtained using reactive slug flowmicroextraction nanoESI with hydroxylamine as the reagent. 10 μL of 8ng/mL epitestosterone (FIG. 5 panel A), 5 ng/mL 5α-androstan-3β,17β-diol-16-one (FIG. 5 panel B), 5 ng/mL 6-dehydrocholestenone (FIG. 5panel C), and 5 ng/mL stigmastadienone (FIG. 5 panel D) in syntheticurine. 5 μL aqueous solution containing 0.1% acetic acid and 10%hydroxylamine were added as the reagent phase. 5 μL ethyl acetate wasused as the extraction phase.

FIG. 6 shows an exemplary three-phase fluid system. Sample analysisusing immiscible three phase SFME: Sample phase is of high polarity andthe solvent of relatively high polarity, such as H₂O and acetonitrile,is used as extraction solvent. Solvent of relatively low polarity whichis immiscible with sample and extraction solvent is plugged between themand keep them separated. A hydrophobic tubing rather than a glass tubingis used in this case to ensure the isolation. A push and pull force isapplied to induce the slug flow movement for extraction. After theextraction, the extract can be either directly or indirectly analyzed bynanoESI, or stored for further operations.

FIG. 7 shows microextraction for analyzing chemicals in low-polaritysamples. Solvents of relatively high polarity can be used for extractionand spray ionization.

FIG. 8 panels A-B shows analysis of vegetable oil using systems andmethods of the invention. A mixture of water and methanol was used asthe extraction solvent. FIG. 8 panel A is an MS spectrum showing thatdiacylglycerol and triacylglycerol species were observed in the MSspectrum in positive mode. FIG. 8 panel B is an MS spectrum showing thatdifferent fatty acids were observed in the MS spectrum acquired innegative mode.

FIG. 9 panels A-B show analysis of 100 ng/mL phenylalanine (165 Da,logP=−1.38) in urine using the 3-phase SFME (FIG. 6). MS/MS spectrum ofthe molecular ion were collected. FIG. 9 panel A shows an MS spectrum ofa 5 μL sample that was 10× diluted using methanol as reduction of matrixand sprayed directly by nanoESI. FIG. 9 panel B shows an MS spectrum ofa 5 μL sample that was processed by 3-phase SFME with hexane/H₂O: MeOH(1:1) as the bridging/extraction solvent, and then analyzed by nanoESI.

FIG. 10 show analysis of 50 ng/mL amitriptyline in bovine whole bloodusing a fused silica capillary.

FIG. 11 panel A shows in-capillary sample extraction using the slug flowmicro-extraction. FIG. 11 panel B shows subsequent MS analysis withnanoESI. MS/MS spectra. FIG. 11 panel C shows analysis of 10 ng mL⁻¹methamphetamine in 5 μL urine. FIG. 11 panel D shows analysis of 50 ngmL⁻¹ benzoylecgonine in 5 μL urine. FIG. 11 panel E shows impact of thenumber of SFME cycles on the extraction of the analytes, intensities ofthe MS/MS product ions monitored for methamphetamine (m/z 150→91),nicotine (m/z 163→130) and benzoylecgonine (m/z 290→168), each at 50ng/mL in urine samples. 2 kV used for nanoESI.

FIG. 12 panels A-B show that the movements of liquid plugs inside thecapillary could be created in two ways. FIG. 12 panel A shows gentlytilting capillary up and down. FIG. 12 panel B shows adding apush-and-pull force by air pressure through a pipette. The pipettingvolume was set to 10 μL for this purpose.

FIG. 13 panels A-B show spectra recorded for direct MS/MS analysis. FIG.13 panels A shows 1 ng mL⁻¹ verapamil in 5 μL undiluted human pooledblood. FIG. 13 panels B shows the endogenous creatinine contained inhuman whole blood.

FIG. 14A shows derivations for calculating of concentrations atequilibrium.

FIG. 14B shows derivations for calculating internal standard (IS)incorporation concentrations at equilibrium after SFME.

FIG. 15 shows quantitative analysis of whole blood spiked withmethamphetamine (1-100 ng mL⁻¹). The blood samples were diluted 10 timesfor decrease in viscosity. Methamphetamine-d8 (2 ng mL⁻¹) in extractionsolvent ethyl acetate.

FIG. 16 panel A shows reactive SFME-nanoESI with a reagent plug injectedbetween the biofluid sample and the extraction solvent. MS/MS spectraof. FIG. 16 panel B shows direct SFME-nanoESI analysis of 200 ng mL-1epitestosterone in synthetic urine. 5 μL water containing 50 mM hydroxylamine was used for the reagent liquid plug. FIG. 16 panel C showsreactive SFME-nanoESI analysis of 200 ng mL-1 epitestosterone insynthetic urine. 5 μL water containing 50 mM hydroxyl amine was used forthe reagent liquid plug.

FIG. 17 panel A shows the reaction scheme of the enzymatic conversion ofacetylthiocholine (ATCh) to thiocholine (TCh) catalyzed bycholinesterase (ChE). FIG. 17 panel B shows progression curve of ATChdigestion determined by SFME-nanoESI. The incubation was over 30 minuteand catalyzed by blood cholinesterase (ChE) in room temperature.Acetylthiocholine iodide standard was added into human whole blood witha final concentration of 1.8 mg/mL before incubation. The intensityratios of product ions from thiocholine (m/z 102→61) and enzymesubstrate (m/z 162→103) were monitored using MRM. FIG. 17 panel C showsevaluation of blood ChE with different levels of enzyme inhibition. TheChE activity in blood sample was determined by SFME-nanoESI after 5 minincubation.

FIG. 18 panel A shows SFME-nanoESI-MS spectra recorded for SFME-nanoESIMS analysis of samples (54, each) taken immediately (top) and 60 min(bottom) after the mixing of the acetylthiocholine into the dilutedblood. FIG. 18 panel B shows investigation of the impact on enzymaticactivity by the extraction solvents. For testing of a particularsolvent, 5 μL blood sample (diluted) was injected into the capillarywith the extraction solvent plug immediately after the mixing theacetylthiocholine into the blood. After the 5 min incubation incapillary, the TCh/ATch ratio was measured using SFME nanoESI MS. Forthe control, 5 min after the mixing acetylthiocholine into the dilutedblood, the sample was taken and analyzed immediately using SFME nanoESIMS with ethyl acetate as the extraction phase. Spray voltage of 1.5 kVwas used for nanoESI.

FIG. 19 shows comparison of different organic solvents for extractionphase used in SFME-nanoESI. For each test, the whole bovine blood spikedwith 5 ng mL⁻¹ methamphetamine was diluted 10 times with water; 5 μLsample was then used to prepare the sample plug and 5 μL of one organicsolvent was used for the extraction plug. SRM (single reactionmonitoring) with a transition m/z 150→91 was used to record theintensity of product ion m/z 91 from protonated methamphetamine m/z 150,while the spray voltage is varied.

FIG. 20 panels A-D shows MS/MS analysis of drugs or steroids of lowconcentrations in 5 μL biological samples (undiluted). FIG. 20 panel Ashows 0.5 ng mL⁻¹ methamphetamine in bovine whole blood. FIG. 20 panel Bshows 0.5 ng mL⁻¹ amitriptyline in bovine whole blood. FIG. 20 panel Cshows 0.8 ng mL⁻¹ verapamil in bovine whole blood. FIG. 20 panel D shows9 ng mL⁻¹ epitestosterone in synthetic urine using reactive SFME.

FIG. 21 shows derivatization and MS/MS fragmentation pathways ofepitestosterone as well as a Table that provides a summary of the targetions for MS/MS analysis of steroid using reactive SFME-nanoESI.

FIG. 22 is a table showing LODs obtained with SLME nanoESI and cut-offconcentrations for detection or monitoring.

FIG. 23 shows an embodiment of SFME used for sampling of a bulk liquidsample (larger volume liquid sample, e.g., greater than 100 μl, in thisexample, a few milliliters).

FIG. 24A shows a comparison of analyses of 5 ng/mL methamphetamine in 10μL and 2 mL urine samples, 10 μL ethyl acetate used as extractionsolvent. FIG. 24B shows a comparison of analyses of 50 μg/mL verapamilin 5 μL and 2 mL urine samples, 5 μL ethyl acetate used as extractionsolvent.

FIG. 25 is a graph showing the

$\frac{I_{a}}{I_{IS}}$

ratio measured with variations in volumes for the extraction solventwith Internal Standard (ethyl acetate containing 40 ng/mL amitraz (IS))and urine samples containing cotinine as the analyte (A) at 300 ng/mL.Volume of ethyl acetate randomly selected between 6-9 μL.

FIG. 26 shows a calibration curve established with

$\frac{I_{a}}{I_{IS}}$

ratios measured for a series urine samples of different volumes andcontinuing cotinine at different concentrations. 10 μL ethyl acetatecontinuing verapamil (log P=3.8) at 5 ng/mL was used as the extractionsolvent for each SFME.

FIG. 27A is a schematic illustrating 3-phase slug flow micro extraction.FIG. 27B is an MS/MS spectrum recorded using TSQ for 45 ng/mL TFV-DP in104, whole blood lysate.

DETAILED DESCRIPTION

The invention provides systems and methods for slug flow microextraction(SFME), optionally followed by ionization of the extracted analyte forrapid analysis of samples. Systems and methods of the invention areuseful for analysis of analytes in any commercial or research field,such as the biomedical field, the pharmaceutical field, the food safetyfield and environmental fields.

In certain embodiments, the invention provides a system for analyzing ananalyte in a sample. FIG. 1A provides an exemplary embodiment of asystem of the invention. The system includes an ionization probe and amass spectrometer. The ionization probe includes a hollow body thatincludes a distal tip. Numerous different types of hollow bodies can beenvisioned by the skilled artisan, and all will work with systems of theinvention. The hollow body can have a distal tip for ejecting a spray ofa solvent that is loaded into the probe. An exemplary hollow body is anano-ESI probe capillary with a distal tip. Exemplary nano-ESI probesare described for example in each of Karas et al. (Fresenius J AnalChem. 366(6-7):669-76, 2000) and El-Faramawy et al. (J Am Soc MassSpectrom, 16:1702-1707, 2005), the content of each of which isincorporated by reference herein in its entirety. Nano-ESI needles arecommercially available from Proxeon Biosystems (Odense, Denmark) and NewObjective Inc (Woburn, Mass.). In other embodiments, the system mayinclude a sample cartridge containing one or more spray tips and one ormore electrodes.

An exemplary hollow body is a glass borosilicate capillary of 0.86 mminner diameter with a pulled tip. The tip will typically have a diameterfrom about 2 μm to about 50 μm. Plastic and rubber tubing can also beused for the hollow body. For example, the hollow body can be composedof PEEK tubing (polyether ether ketone polymer tubing) or TEFLON tubing(polytetrafluoroethylene (PTFE) polymer tubing) or TYGON tubing(flexible tubing consisting of a variety of base materials).

An exemplary hollow body is a fused silica capillary of 0.5 mm or 0.25mm inner diameter, with or without a pulled tip.

As shown in FIG. 1A, the hollow body is loaded with at least twoimmiscible fluids, such as a solvent and a sample that is immisciblewith the solvent, and an extraction is conducted within hollow body ofthe probe. Those aspects of the invention will be discussed in greaterdetail below. In certain embodiments, in order to conduct the extractionwithin the probe body, the body should be devoid of any other material.For example, there are no substrates (e.g., porous substrates, such aspaper substrates), filters, beads, gels, or other substances disposedwithin the body. Rather, the body remains completely empty of othersubstances in order to receive the immiscible fluids that will beinvolved in the extraction. In certain embodiments, magnetic beads areadded into the sample and solvent plugs and an alternating magneticfield is applied to induce the movements of the magnetic beads insidethe liquid plugs, thereby to facilitate the turbulents inside each plugfor transporting the analytes to and from the liquid-liquid interface.

In certain embodiments, an inner surface of the body is coated to adjustthe hydrophobicity of the inner surface of the body. Hydrophobic regionsmay be coated onto the surface using known techniques, such as by usingphotolithography, printing methods or plasma treatment. See Martinez etal. (Angew. Chem. Int. Ed. 2007, 46, 1318-1320); Martinez et al. (Proc.Natl Acad. Sci. USA 2008, 105, 19606-19611); Abe et al. (Anal. Chem.2008, 80, 6928-6934); Bruzewicz et al. (Anal. Chem. 2008, 80,3387-3392); Martinez et al. (Lab Chip 2008, 8, 2146-2150); and Li et al.(Anal. Chem. 2008, 80, 9131-9134), the content of each of which isincorporated by reference herein in its entirety. In certainembodiments, the body is prepared to have a uniform hydrophobicity. Inother embodiments, the body can be prepared to have numerous differentregions, each have different hydrophobicity, which can be based on thetype of liquid that will fill that region of the body. For example, aregion of the body that will receive an oil based sample can be treatedto be more hydrophobic than a region of the body that will receive awater and methanol based solvent.

In certain embodiments, the hollow body is configured such that there isno electrode disposed on a surface of the body. Instead, an electrode isat least partially disposed within the hollow body. As shown in FIG. 1A,the electrode can be a metal wire that extends into the hollow body. Anymetal typically used for electrodes can be used for the metal electrode.That metal wire is connected to a voltage source, such as a high voltagesource. The length of the metal wire shown in FIG. 1A is only exemplary.The metal wire can extend any length into the hollow body. The metalwire can extend to a distal end of the hollow body, as shown in FIG. 1A.Alternatively, the metal wire can be much shorter than shown in FIG. 1A,extending not as far into the body. The amount of solvent added to thehollow body will determine the length of the metal wire, as the wireshould extend far enough into the body to interact with the solvent thathas been added to the body.

As shown in FIG. 1A, the metal wire may be coaxially disposed within thehollow body, although this is not required. Typically, the metal wiredoes not touch as the walls of the hollow body, as shown in FIG. 1A. Themetal wire electrode and its coupling can be removably or permanentlyattached to the hollow body. As shown in FIG. 1A, the metal wireelectrode and its coupling are removably attached to the hollow body.That allows the proximal end of the hollow body to act as a port forintroduction of fluids into the body. In such as embodiment, the metalwire electrode and its coupling is removed from the hollow body, leavingan opening through which fluids are introduced into the body. Onceintroduced, the metal wire electrode and its coupling are attached tothe hollow body, sealing the hollow body.

In other embodiments, the attachment is a permanent attachment and oneor more separate fluid ports along the body are used to introduce thefluids to the hollow body. Even if the attachment of the metal wireelectrode and its coupling to the hollow body is a removable attachment,the hollow body can still include one or more separate ports along thebody to introduce the fluids to the hollow body.

As shown in FIG. 1A, the introduction of high voltage to the liquidwithin the hollow body ejects the liquid from the distal tip of thehollow body in the form of a spray. An inlet of a mass spectrometer isoperably located to receive the liquid ejected from the probe. Thatdistance is typically less than 10 mm, however any distance that allowsa signal from the sample to be generated within the mass spectrometer issuitable. That distance can by determined by the skilled artisan bysimply adjusting the spacing between the probe and the inlet of the massspectrometer and monitoring the read-out generated by the massspectrometer.

In other embodiments, the outside wall of the pulled tip can be coatedwith metal. The high voltage can be applied through the metal coatingfor the spray ionization. Any type of mass spectrometer known in the artcan be used with proves of the invention.

For example, the mass spectrometer can be a standard bench-top massspectrometer. In other embodiments, the mass spectrometer is a miniaturemass spectrometer. An exemplary miniature mass spectrometer isdescribed, for example in Gao et al. (Z. Anal. Chem. 2006, 78,5994-6002), the content of which is incorporated by reference herein inits entirety. In comparison with the pumping system used for lab-scaleinstruments with thousands watts of power, miniature mass spectrometersgenerally have smaller pumping systems, such as a 18 W pumping systemwith only a 5 L/min (0.3 m3/hr) diaphragm pump and a 11 L/s turbo pumpfor the system described in Gao et al. Other exemplary miniature massspectrometers are described for example in Gao et al. (Anal. Chem.,80:7198-7205, 2008), Hou et al. (Anal. Chem., 83:1857-1861, 2011), andSokol et al. (Int. J. Mass Spectrom., 2011, 306, 187-195), the contentof each of which is incorporated herein by reference in its entirety.Miniature mass spectrometers are also described, for example in Xu etal. (JALA, 2010, 15, 433 -439); Ouyang et al. (Anal. Chem., 2009, 81,2421-2425); Ouyang et al. (Ann. Rev. Anal. Chem., 2009, 2, 187-214);Sanders et al. (Euro. J. Mass Spectrom., 2009, 16, 11-20); Gao et al.(Anal. Chem., 2006, 78(17), 5994 -6002); Mulligan et al. (Chem.Com.,2006, 1709-1711); and Fico et al. (Anal. Chem., 2007, 79, 8076 -8082),the content of each of which is incorporated herein by reference in itsentirety.

In certain embodiments, the mass spectrometer inlet is located remotefrom the ionization probe and an ion transfer member is used to transferover longer distances. Exemplary ion transfer members are described forexample in Ouyang et al. (U.S. Pat. No. 8,410,431), the content of whichis incorporated by reference herein in its entirety.

In certain embodiments, the ionization probes of the invention operatewithout pneumatic assistance. That is, with probes of the invention,pneumatic assistance is not required to transport an analyte; rather, avoltage is simply applied to the substrate that is held in front of amass spectrometer. However, in certain embodiments, nebulizing gas maybe used with systems of the invention to assist with desolvation. Thenebulizing gas may either be pulsed or provided as a continuous flow. Inother embodiments, a gas generating device is operably coupled to theprobe such that it can inject a gas into the hollow body to push thesample and solvent to a distal tip of the probe. The gas will typicallybe an inert gas, such as nitrogen or argon, but can also be air.

In certain embodiments, the ionization probe is kept discrete (i.e.,separate or disconnected) from a flow of solvent, such as a continuousflow of solvent. Instead, discrete amounts of solvent and sample areintroduced into the hollow body of the probe. The probe is thenconnected to a voltage source to produce ions of the sample which aresubsequently mass analyzed. The sample is transported through the hollowbody without the need of a separate solvent flow. As previouslymentioned, pneumatic assistance is not required to transport theanalyte; rather, a voltage is simply applied to the solvent in the probethat includes the extracted analyte that is held in front of a massspectrometer.

FIG. 1B shows an exemplary method of use for systems of the invention.In certain embodiments, such methods involve introducing a solvent intoa hollow body including a distal tip. A sample is also introduced intothe hollow body. The solvent is immiscible with the sample and extractsat least one analyte from the sample into the solvent. A voltage isapplied to the solvent including the extracted analyte in the hollowbody so that the analyte is expelled from the distal tip of the body,thereby generating ions of the analyte. Those expelled ions are thenanalyzed.

FIG. 1B shows two immiscible phases, the liquid sample and the organicsolvent, injected adjacently in a capillary with a pulled tip. Given thedifferent polarity of the different phases, one or more analytes willmove from the sample into the solvent (extraction of analytes from thesample into the solvent). That extraction process can be facilitated bycausing the liquid phases to move back and forth in the capillary, suchas by tilting the capillary or applying gas pressure, to facilitate themicroextraction. The liquid phases may then be pushed with theextraction phase reaching the pulled tip of the capillary, by applying agas pressure (from a gas generating device operably coupled to theprobe), and a wire electrode is inserted into the extraction solvent toapply a DC voltage for nanoESI. The voltage causes the solvent to beexpelled from the distal tip of the hollow body as a spray which reachesthe inlet of the mass spectrometer.

Methods of the invention can be used with any type of sample, such asorganic or non-organic, biological or non-biological, etc. In certainembodiments, the sample is derived from a biological tissue or is abiological fluid, such as blood, urine, saliva, or spinal cord fluid.The sample may include an analyte of interest to be analyzed. Thatanalyte can be native to the sample or may have been introduced into thesample. Exemplary analytes include therapeutic drugs, drugs of abuse andother biomarkers. The Examples herein show that effective suppression ofthe matrix effect was achieved for therapeutic drugs, drugs of abuse andother biomarkers. In certain embodiments, systems and methods of theinvention can be used for direct analysis of the biofluid samples orliquid samples.

The solvent may be any solvent so long as it is immiscible with thesample and works for both extraction and ionization of the sample.Typically, the chosen solvent will depend on the sample to be analyzedand/or the analyte of interest believed to be in the sample (FIG. 19). Afactor to be considered is the polarity of the solvent. In the 2-phaseextraction system, ideally the solvent has a different polarity then thesample and/or the analyte of interest believed to be in the sample. Forexample, an aqueous sample will typically have a high polarity, andtherefore a good choice of solvent would be an organic solvent with alow polarity (e.g., methanol or ethyl acetate or mixtures that includethose solvents e.g., water/methanol mixtures or water/ethyl acetatemixtures). An oil sample will typically have a low polarity, andtherefore a good choice of solvent would be a solvent with a higherpolarity, such as a water/methanol mixture. The skilled artisan will beable to determine the proper solvent to use based on the sample to beanalyzed.

Another consideration of the solvent is that in addition to being goodfor an extraction of an analyte from a sample, it can also be used toionize the sample. That is, the solvent can be compatible for both theextraction and the ionization of the extracted analyte. As illustratedin the Example, methanol and ethyl acetate work well for extraction ofanalytes as well as for ionization of analytes, while chloroform workswell for extraction but not for ionization of analytes. Typically, asolvent that is compatible with electrospray ionization can possibly beused with systems and methods of the invention, so long that solvent isalso immiscible with the sample and is able to extract an analyte fromthe sample. The skilled artisan having experience in mass spectrometrywill know particular solvents that are compatible with electrosprayionization.

Methods of the invention can also involve real-time chemical reactionsthat can be used for improving the overall analysis efficiency of thetarget analytes. To perform such a derivation, a solution containing anagent that imparts a charged function group to the analyte is introducedto the hollow body. That solution is typically introduced between thesolvent and the sample. The agent in the solution interacts with theanalytes in the sample and imparts a charged functional group to thesample, allowing for the ionization of the analyte.

In certain embodiments, more than one analyte (e.g., a plurality ofanalytes) is extracted from the sample and into the solvent. Theplurality of analytes can be extracted at the same time. Alternatively,the analytes are differentially extracted into the solvent, typicallybased on the polarity of the analyte and the polarity of the solvent.

While methods of the invention have been discussed using two immisciblefluid, the systems and methods of the invention are not limited to theuse of two fluids. Any number of fluids can be used with systems andmethods of the invention, such as three fluids, four fluids, fivefluids, etc. In certain embodiments, a three fluid system is used. Insuch embodiments, two miscible fluids are separated by an immisciblefluid. An exemplary three fluid system is shown in FIG. 6. Thepolarities of the Sample-Solvent Bridge-Extraction/Spray Solvent can behigh-low-high or low-high-low. A capillary surface with properhydrophobicity can be selected to stabilize the solvent bridge, whichseparates the sample phase and the extraction solvent phase of similarpolarities (which means they are miscible). As an example, a urinesample plug and a methanol/water plug for extraction can be separated byethyl acetate or hexane, and a Teflon capillary with hydrophobic surfacecan be used.

In certain embodiments, systems and methods of the invention can also beused for preparing samples that will be analyzed later. The extractionsolvent can be stored as the liquid sample or deposited on papersubstrate or MALDI plate to prepare the dried sample spots. The internalstandard can be incorporated in to the dried sample spots during theSFME process. The target analytes can be chemical modified during theSFME process.

In other embodiments, the hollow body does not require a distal tipbecause the extraction capillary is not used as an ionization probe. Insuch embodiments, the extraction is simply conducted as described abovein a capillary. After the extraction is completed, the solventcontaining the extracted analyte is removed from the capillary and isthen analyzed using any method known in the art. For example, thesolvent containing the extracted analyte may be loaded into a separateionization probe and then analyzed by mass spectrometry, such as shownin FIG. 6. In other embodiments, the analyte is analyzed in a differentmanner, such as any spectroscopy technique or other assay known in theart.

In other embodiments, the invention provides methods that are analyzinglarger volume samples. Larger volume samples are samples greater than100 μl, as opposed to small volume samples, which are samples less than100 μl. Exemplary large volume samples can range from the microliterrange (e.g., greater than 100 μl) into the milliliter range, and intothe liter range and above.

Typically, larger volume samples are contained in a vessel, such as astandard vessel for holding a biological sample, such as a VACUTAINER(blood collection tube, commercially available from BD). Other vessels,such as standard laboratory vessels (beakers, flasks, etc.) can be usedto hold larger volume samples.

Larger volume samples typically include urine samples or otherbiological fluids, such as blood. Generally, a body fluid refers to aliquid material derived from, for example, a human or other mammal. Suchbody fluids include, but are not limited to, mucus, blood, plasma,serum, serum derivatives, bile, phlegm, saliva, sweat, amniotic fluid,mammary fluid, urine, sputum, and cerebrospinal fluid (CSF), such aslumbar or ventricular CSF. A body fluid may also be a fine needleaspirate. A body fluid also may be media containing cells or biologicalmaterial. Larger volume samples can also be environmental samples, suchas river water, soil, etc.

In addition to native components of the sample, the biological orenvironmental samples can include a non-native biological agent that canbe analyzed by methods of the invention. Exemplary environmental samplesinclude a water sample or a soil sample. In certain embodiments, abiological agent include all genuses and species of bacteria and fungi,including, for example, all spherical, rod-shaped and spiral bacteria.Exemplary bacteria are stapylococci (e.g., Staphylococcus epidermidisand Staphylococcus aureus), Enterrococcus faecalis, Pseudomonasaeruginosa, Escherichia coli, other gram-positive bacteria, andgram-negative bacilli. An exemplary fungus is Candida albicans. Abiological agent also includes toxins secreted by bacteria or fungi. Forexample, E. coli secretes Shiga-like toxin (Zhao et al., AntimicrobialAgents and Chemotherapy, 1522-1528, 2002) and C. Difficile secretesExotoxin B (Sifferta et al. Microbes & Infection, 1159-1162, 1999). Abiological agent can also include an allergen. An allergen is anonparasitic antigen capable of stimulating an immune response in asubject. Allergens can include plant pollen or dust mite excretion.

The extraction solvent may be suitable for extracting nucleic acid fromthe biological agent. SDS-based extraction may be suitable. See forexample, Bhat et al. (U.S. Pat. No. 7,208,654), the contents of whichare incorporated by reference herein in their entirety.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Micro-Extraction Protocol

Systems and methods of the invention were used to analyze 10 μL urinesamples containing benzoylecgonine, nicotine or methamphetamine, withLODs better than 1 ng/mL achieved (FIG. 2 panels A-C). Chemicalequilibrium was reached faster at higher tilting rates (˜30/minute).Significant improvement of signal was observed even for analytes withrelatively low patrician coefficient for the extraction solvent, due toan effective suppression of the matrix effect. Different solvents weretested for the extraction. Nonpolar solvents such as chloroform werefound to be efficient for extraction but relatively poor for subsequentionization. An on-line injection of methanol could be used to facilitatethe direct ionization of the analytes extracted into these solvents.Ethyl acetate, however, was found to be effective for both extractionand ionization, such as by nanoESI. Various methods have also beenexplored for incorporation of internal standards for quantitation whilemaintaining the operation procedure simple. A calibration of nicotinewith good linearity (R2=0.99) has been obtained (FIG. 2 panel B).

Example 2 Real-Time Derivatization

Methods of the invention can also involve real-time chemical reactionsthat can be used for improving the overall analysis efficiency of thetarget analytes. This is exemplified by the analysis of a steroid inurine. The efficiency was expected to be high for extracting steroidsfrom the urine; however, steroids are difficult to ionize by sprayionization. A real-time derivatization was performed for SFME-nanoESI byinjecting 3 μL aqueous solution with 5% hydroxylamine between theextraction solvent (ethyl acetate) and the urine sample (FIGS. 3A-B).The reactant solution mixed quickly with the sample and the steroidswere derivatized with a charged function group while being extractedinto the organic phase. The signals in MS spectra were improved bymultiple orders of magnitudes. LODs of 0.2, 0.7, 0.6, 0.8 ng/mL wereobtained for 5α-androstan-3β, 17β-diol-16-one, epitestosterone,4,6-cholestadien-3-one, and stigmastadienone, respectively, in urinesamples of amounts below 10 mL.

Scheme 1. Reaction between hydroxylamine and the carbonyl groups onsteroids

FIG. 5 panels A-D show MS/MS spectra obtained using reactive slug flowmicroextraction nanoESI with hydroxylamine as the reagent. 10 μL of 8ng/mL epitestosterone (FIG. 5 panel A), 5 ng/mL 5α-androstan-3β,17β-diol-16-one (FIG. 5 panel B), 5 ng/mL 6-dehydrocholestenone (FIG. 5panel C), and 5 ng/mL stigmastadienone (FIG. 5 panel D) in syntheticurine. 5 μL aqueous solution containing 0.1% acetic acid and 10%hydroxylamine were added as the reagent phase. 5 μL ethyl acetate wasused as the extraction phase.

The analysis of the samples described above would otherwise need to beanalyzed using traditional lab procedures using sample extraction,liquid chromatography, and mass analysis using electrospray ionizationor atmospheric pressure chemical ionization. The sample amounts requiredare significantly larger (˜1 mL).

Example 3 Direct Analysis of Biological Fluids with Low Viscosity

Biological samples such as urine were directly analyzed using the SFMEnanoESI. FIG. 2 panels A-C. Calibration curves for quantitation ofmethamphetamine (FIG. 2 panel A), nicotine (FIG. 2 panel B), andbenzoylecgonine (FIG. 2 panel C) in synthetic urine samples. 10 μLsynthetic urine containing the drugs and internal standards were used assamples for the measurement. 5 μL ethyl acetate (EA) was used as theextraction phase for extraction, purification and spray. Internalstandards: methamphetamine-d8 at 0.8 ng/mL, nicotine-d32 at ng/mL,benzoylecgonine-d3 at 1 ng/mL. The single reaction monitoring (SRM)transitions used: methamphetamine m/z 150→91, methamphetamine-d8 m/z158→93; nicotine 163→130, nicotine-d3 m/z 166→130; benzoylecgonine m/z290→168, benzoylecgonine-d3 m/z 293→171. Partition coefficients:LogP_(methamphetamine)=2.07; LogP_(nicotine)=1.17,LogP_(benzoylecgonine)=−0.59.

The matrix effect due to high concentration salts were minimized. GoodLODs were obtained for drugs of abuse, even for benzoyecgonine withrelatively low partition coefficient for the extraction phase. Thepartition coefficient (LogP) is defined as:LogP=log([solute]_(octanol)/[solute]_(water)), which represents thedifferential solubility of an un-ionized compound in an organic phasesuch as octanol immiscible with the aqueous phase at equilibrium.

Example 4 Direct Analysis of Viscous Biofluids

For viscous biofluid samples, dilution of the sample was applied toallow the operation with systems and methods of the invention. As anexample, blood samples containing drugs were diluted 10 times beforeanalysis by SFME nanoESI. The data in FIG. 4 panels A-B show thatmethods of the invention were able to analyze analytes from a bloodsample. FIG. 4 panels A-B show MS/MS spectra obtained using slug flowmicroextraction nanoESI. Bovine blood samples, each containing 40 ng/mLnicotine (FIG. 4 panel A) and 40 ng/mL methamphetamine (FIG. 4 panel B)were diluted 10 times with water and then analyzed using SFME nanoESI.10 μL of diluted sample, 5 μL ethyl acetate used.

Example 5 Summary of the Analytical Performance

TABLE 1 Limits of detection of chemicals in synthetic urine and bloodsamples achieved using slug flow microextraction nanoESI Sample volumeLOD Compound Matrix Derivatization (μL) (ng/mL) MethamphetamineSynthetic NA 5 0.03 urine Bovine blood NA 5 <40 Nicotine Synthetic NA 50.1 urine Bovine blood NA 5 <40 Benzoylecgonine Synthetic NA 5 0.08urine Epitestosterone Synthetic hydroxylamine 5 0.7 urine 6-dehydro-Synthetic hydroxylamine 5 0.6 cholestenone urine 5α-androstan- Synthetichydroxylamine 5 0.2 3β,17β-diol-16-one urine Stigmastadienone Synthetichydroxylamine 5 0.8 urine

Example 6 Direct Analysis of Oil Samples

The Examples above show that the drug compounds in aqueous samples ofhigh polarities, such as blood or urine, were extracted to organicsolvents of low polarity. The systems and methods of the invention canalso be applied by extracting analytes from samples of low polaritysamples, such as oil, to the extraction solvent of high polarity, suchas the water/methanol solvent as shown in FIG. 7. The results are shownin FIG. 8, which shows analysis of vegetable oil using systems andmethods of the invention. A mixture of water and methanol was used asthe extraction solvent. FIG. 8 panel A is an MS spectrum showing thatdiacylglycerol and triacylglycerol species were observed in the MSspectrum in positive mode. FIG. 8 panel B is an MS spectrum showing thatdifferent fatty acids were observed in the MS spectrum acquired innegative mode.

Example 7 Three-Phase Methods

A three-phase method can be performed as exemplified in FIG. 6. Thepolarities of the Sample-Solvent Bridge-Extraction/Spray Solvent can behigh-low-high or low-high-low. A capillary surface with properhydrophobicity can be selected to stabilize the solvent bridge (middlephase), which separates the sample phase and the extraction solventphase of similar polarities (which means they are miscible). As anexample, the urine sample plug and the methanol/water plug forextraction can be separated by ethyl acetate or hexane, and a Tefloncapillary with hydrophobic surface can be used. Analysis ofphenylalanine from urine is shown in FIG. 9 panels A-B. Phenylalanine isof relatively high polarity. The phenylalanine molecules were extractedfrom the urine to H₂O:MeOH (1:1) through the hexane, which separatesthem from the salts in the urine. This is a purification process.

If two-phase methods with urine and hexane are used, phenylalanine is ofrelatively high polarity so the solubility in hexane is relatively lowand the concentration would be low in hexane. Also, hexane is much lessfavorable for spray ionization in comparison with the polar solventssuch as H₂O:MeOH (1:1). The three-phase methods with asample-bridge-spray in an polarity order as high-low-high allow acompound of high polarity to be concentrated into a high polaritysolvent, which is suitable for spray ionization. The subsequent analysisis done by transferring the extraction solvent to a capillary with apulled tip for spray ionization (FIG. 6) or with a direct sprayionization from the capillary as previously described above. Real timechemical derivatization can be applied by adding the reaction reagentsin either or both of the bridge solvent or the extraction/spray solvent.Real time internal standard incorporation can be applied by pre-addingthe internal standards in either of both of the bridge solvent of theextraction/spray solvent.

Example 8 Micro-extraction in a Fused Silica Tubing (i.d. 500 μm)

The SFME sample processing can be done in fused silica tubing of smallerdiameter which are commonly used as liquid line in liquid chromatographysystem (e.g., tubing having an inner diameter of 500 μm or less). Theextraction can be induced by applying a push and pull force on one sideof the tubing. The extract can be either directly analyzed by nanoESI orstored for further operations.

FIG. 10 show analysis of 50 ng/mL amitriptyline in bovine whole blood.MS/MS spectrum of the molecular ion was collected. The blood sample wasfirst 10× diluted using H₂O as a reduction of viscosity. For extraction,5 μL of the diluted sample was processed in a fused silica tubing (i.d.500 μm) using methods of the invention. The extract was then infusedinto a nanoESI emitter and analyzed by nanoESI.

Example 9 Direct Mass Spectrometry Analysis of Biofluid Samples UsingSlug Flow Microextraction NanoESI

Direct mass spectrometry (MS) analysis of biofluids with simpleprocedures represents a key step in translation of MS technologies tothe clinical and point-of-care applications. The current study reportsthe development of a single-step method using slug flow microextractionand nanoESI (electrospray ionization) for MS analysis of organiccompounds in blood and urine. High sensitivity and quantitationprecision have been achieved for analysis of therapeutic and illicitdrugs in 5 μL samples. Real-time chemical derivatization has beenincorporated for analyzing anabolic steroids. The monitoring ofenzymatic functions has also been demonstrated with the cholinesterasein wet blood. The reported work encourages future development ofdisposable cartridges highly functioning with simple operation, inreplacement of traditional complex lab procedures for MS analysis ofbiological samples.

Mass spectrometry (MS) has been demonstrated as a powerful tool forchemical and biological analysis. The high specificity, high sensitivityand high precision in quantitation are achieved traditionally inlaboratory by eliminating the matrix effect through sample extractionand chromatographic separation prior to the MS analysis. The developmentof ambient ionization, especially with the recent demonstration usingthe paper spray, has indicated a promising future for direct MS analysisof high quantitation performance but using highly simplified protocolsconsuming ultra-small amounts of samples. This would be extremelyimportant for the translation of the MS analysis to out-of-labapplications, especially point-of-care (POC) diagnosis. The underlyingprinciple for a successful development along this direction is tominimize the sample consumption and to achieve high efficiency in anintegrated process for the analyte extraction and ionization. Slug flowmicroextraction (SFME) and nanoESl (electrospray ionization) can becombined to perform a one-step analysis of biofluid samples. Excellentsensitivity and high quantitation precision have been obtained withblood and urine samples of only 5 μL. More importantly, the SFME-NanoESImethod demonstrated how to incorporate a variety of different processesusing a simple device, including liquid-liquid extraction, internalstandard (IS) incorporation, chemical derivatization or even enzymaticreactions, which are necessary for a high performance mass analysis.

All the experiments were carried out with a TSQ Quantum Access Max(Thermo Fisher Scientific, San Jose, Calif., USA).The bovine blood waspurchased from Innovative Research Inv. (Novi, Mich., USA). The humanpooled blood for enzymatic reaction study was purchased fromBioreclamationIVT (Baltimore, Md., USA). The synthetic urine waspurchased from CST Technologies (Great Neck, N.Y., USA). The steroidswere purchased from Steraloids Inc. (Newport, R.I., USA). All otherchemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

A disposable glass capillary of 0.8 mm i.d. (FIG. 11 panel A) with apulled tip for nanoESI was used to perform the entire samplingionization process. Two adjacent liquid plugs were formed bysequentially injecting 5 μL organic solvent and 5 μL urine or bloodsample into the capillary. The liquid-liquid extraction of the analytesfrom the biofluid into the organic solvent is expected, but at a fairlylow efficiency due to the small interfacing area. However, theextraction speed could be significantly improved with the slug flowsinduced by the movements of the two liquid plugs, which can befacilitated by tilting the capillary (FIG. 11 panel A and FIG. 12 panelA) or by applying a push-and-pull force through air pressure (FIG. 12panel B). The slug flows is formed due to the friction with thecapillary wall, and the flows inside each plug (FIG. 11 panel A)transfer the analytes to and away from the liquid-liquid interface,therefore significantly improving the extraction efficiency. After theextraction process, the organic solvent plug can be simply pushed to thetip of the capillary; a stainless steel wire was then inserted throughthe biofluid sample to reach the organic solvent plug; a high voltagewas applied to generate the nanoESI for MS analysis (FIG. 11 panel B).The selection of the organic solvent is important. It should beimmiscible with the biofluid samples, have good solubility for thetarget analytes and be suitable for nanoESI. Several organic solventshave been tested (FIG. 19) and ethyl acetate of a weak polarity wasfound to provide the optimal performance for analyzing a broad range ofchemical compounds in urine (FIG. 11 panels C-D) and blood samples (FIG.13 panels A-B).

The extraction process with the slug flows have been shown to be veryefficient, as tested for extracting methamphetamine, nicotine andbenzoylecgonine (a main metabolite of cocaine) from urine samples. Theequilibrium was reached after tilting the capillary 5 times (FIG. 11panel E and FIG. 20 panels A-D). Limits of detection (LODs) as good as0.05 ng/mL for verapamil, have been obtained for the whole blood samplesusing SFME-nanoESI (Table 2).

TABLE 2 Limits of detection (LODs) for analytes in urine and/or wholeSample Volume LOD Analyte Sample Derivatization (μL) (ng/mL)Methamphetamine Urine NA 5 0.03 Blood NA 5 0.1 Benzoylecgonine LOD UrineNA 5 0.1 Blood NA 5 1 Verapamil Blood NA 5 0.05 Amitriptyline Blood NA 50.08 Epitestosterone Urine Hydroxyl-amine 5 0.7 6-Dehydro- UrineHydroxyl-amine 5 0.6 cholestenone 5α-Androstan- Urine Hydroxyl-amine 50.2 3β,17β-Diol-16-one Stigmastadienone Urine Hydroxyl-amine 5 0.8Fewer extraction cycles were needed for reaching equilibrium if theblood samples were diluted to reduce the viscosity. The distribution ofthe analyte between the sample and extraction phase can be relativelyestimated by the partitioning coefficient (logP, see FIG. 14). Formethamphetamine with a logP of 2.1, its concentration in the organicextraction solvent can be 100 times higher than in the urine sampleafter SFME, which certainly explains the good LOD of 0.03 ng/mL achievedwith urine samples (Table 1). The logP value for benzoylecgonine is−0.6, which means it has higher solubility in urine than in organicsolvents and the extraction into ethyl acetate was a dilution process;however, an LOD of 0.08 ng/mL was achieved regardless. This indicatesthat the limiting factor in the detection of the benzoylecgonine in rawurine samples might not be the absolute amount or concentration of thebenzoylecgonine, but the interference by the matrix effects, such as theionization suppression due to the high concentrations of salts in theurine sample. An efficient separation of the benzoylecgonine from thesalts was achieved in the SFME process. Even with a lowerbenzoylecgonine concentration in the extraction phase, the ionizationefficiency and the overall sensitivity of the analysis were improvedsignificantly.

In addition to the sensitivity, adequate precision in quantitation isoften mandatory for clinical and POC applications. Simple means foraccurate incorporation of internal standard are important but can bechallenging for samples of small volumes taken by minimally invasivemethods. Using the SFME-nanoESI, the IS compounds could be spiked in theextraction phase (FIG. 15) and subsequently mixed with the analyteduring the slug flow extraction process. This method was tested forquantitation of methamphetamine in bovine blood samples withmethamphetamine-d8 as the IS spiked in ethyl acetate at 2 ng/mL. Theblood samples were diluted 10 times and then analyzed using theSFME-nanoESI and MRM analysis (transitions m/z 150 to 91 and m/z 158 to94 for the analyte and IS, respectively) (FIG. 15, inset). The measuredanalyte-to-IS ratios (A/IS) are plotted as a function of the originalanalyte concentration in blood as shown in FIG. 15. A good linearity wasobtained, which is governed by the partitioning process (see derivationin Supporting Information). RSDs better than 10% were obtained forsamples of concentrations higher than 10 ng/mL.

Chemical derivatization is an effective way of altering the propertiesof the target analytes to improve the efficiency of separation orionization for MS analysis. For example, the steroids in urine or bloodsamples are expected to be well extracted into an organic phase usingthe SFME; however, the efficiency for the subsequent ionization bynanoESI would be low due to the low proton affinity of the steroidmolecules. The reaction with hydroxyl amine has previously been provedto be effective in improving the ionization efficiency of the steroids,and thereby was used in this study as an example. An additional liquidplug of 5 μL water containing 50 mM hydroxyl amine was injected betweenthe 5 μL ethyl acetate and 5 μL urine sample spiked with 200 ng ml⁻¹epitestosterone (FIG. 16 panel A). With 5 SFME cycles, the hydroxylamine solution mixed well with the urine sample. The MS/MS analysis ofthe reaction product m/z 304 produced spectra of significantly improvedsignal-to-noise ratios (S/Ns) (FIG. 16 panels

B-C and FIG. 21). The reactive SFME-nanoESI was applied for analysis ofa series of anabolic steroids in 5 μL urine samples, includingepitestosterone, 6-Dehydrocholestenone, 5α-Androstan-3β, 17β-Diol-16-oneand stigmastadienone, with LODs of 0.7, 0.6, 0.2 and 0.8 ng/mL obtained,respectively (Table 1 and FIG. 22).

Using the liquid-liquid extraction process with SFME, the analysis cannow be performed directly with wet blood samples. This provides anopportunity for probing the chemical and biological properties that onlyexist with the original liquid samples. For instance, the enzymaticfunctions of the proteins are typically quenched in the dried bloodspots or after the traditional lab procedure for sample extraction.SFME-nanoESI was applied for monitoring the enzymatic activity ofcholinesterase (ChE) in whole blood samples. The ChE facilitates theenzymatic conversion of acetylthiocholine (ATCh) to thiocholine (TCh)(FIG. 17, panel A). The blood sample was diluted 10 times to slow downthe reaction rate as well as to facilitate the slug flows for SFME. Thesubstrate acetylthiocholine iodine was added into the diluted bloodsample at a concentration of 1.8 mg/mL, and then 5 μL sample was takenimmediately and injected into the capillary with 5 μL extraction phase.The capillary with the sample and the extraction solvent was left inroom temperature 25° C. for incubation. The SFME-nanoESI could beperformed repeatedly on the same sample and the ratio of the substrateATCh and the reaction product TCh could be monitored as a function oftime to characterize the enzymatic activity of the ChE. A potentialproblem in this approach would be the damage to the enzyme function bythe organic solvent. The impact by organic extraction phase wasinvestigated for ethyl acetate and other solvents such as chloroformwith a 5 min incubation. It was found that the reduction of ChE activitydue to the contact with ethyl acetate was minimal but much more severe(more than 60% decrease) with chloroform. A weakly polar solvent likeethyl acetate can better preserve the enzyme structures.

Using ethyl acetate as the extraction solvent, the SFME-nanoESI wasperformed repeatedly over 30 min, with 5 cycles for SFME and 5 s nanoESIat 1500 V for each analysis. The TCh/ACTh ratio is plotted as a functionof time in FIG. 17 panel B, which is characteristic for the enzymaticactivity of the ChE. An enzyme inhibition study was then carried out asa validation of this method. Two ChE inhibitors, donepezil (atherapeutic drug for Alzheimer's disease) and ethion (a neurontoxicant), were spiked separately into blood samples, simulating theenzyme inhibitions at different degrees. The compromised enzymeactivities were then determined using the SFME-nanoESI method with 5 minincubation. In comparison with the blood samples without adding theinhibitors, the deficiencies measured are reported in FIG. 17 panel Cfor blood samples treated with donepezil at 25 ng/mL and 5 μg/mL, andwith ethion at 10 μg/mL. The percent decreases observed are consistentwith the findings reported for previous studies.

In summary, the combination of the slug flow microextraction withnanoESI enabled a high-sensitivity direct analysis of the organiccompounds in biofluids. Multiple types of processes for sampletreatments, which traditionally require complex setups in lab, can nowbe incorporated into a one-step analysis with extremely simplifiedoperation procedure. Since the biofluid samples are directly analyzedwithout being made into dried spots, an efficient liquid-liquidextraction can be designed based on the partitioning properties. Thechemical and biological properties of the wet biofluids can also beretained and characterized thereby. The extraction process can be turnedon and off by controlling the movements of the sample and extractionplugs. This allows an on-line monitoring of the chemical and biologicalreactions in a biofluid sample of only 5 μL. With the increasinginterest in the translation of MS technologies to the clinicalapplications, this development has a profound implication on designingdisposable sample cartridges with adequate function for direct analysis.This could ultimately lead to an elimination of the traditional labprocedures requiring complex setups and expertise. Its implementationwith miniature mass spectrometers would produce a powerful solution forPOC diagnosis.

Example 10 Enzymatic Activity Monitoring by SFME-NanoESI

For initiating an enzymatic reaction, acetylthiocholine (finalconcentration of 1.8 mg mL-1) was added into human blood sample, whichhad been diluted 10 times with phosphate buffered saline (PBS). Forexperiment producing the data for FIG. 17 panel B, 5 μL blood samplewith acetylthiocholine added was loaded into a capillary along with 5 μLextraction solvent. Enzymatic reaction progress was determined byperiodically performing the SFME-nanoESI MS analysis of the substrate(m/z 162) and the reaction product thiocholine (m/z 120) (FIG. 18 panelsA-B). For each SFME-nanoESI MS analysis, the liquid plugs were pushed tolet the extraction solvent reach the capillary tip for spray and thenpulled back after the MS analysis. MRM was performed for measuring theintensities of TCh (m/z 120→61) and ATCh (m/z 162→102). The ratios ofTCh/ATch are used for making the plot in FIG. 17 panel B. Threereplicates were performed for each time point. The standard deviationsare marked with the error bars in the FIG. 17 panel B.

Example 11 Bulk Sampling and Quantification

The slug flow microextraction (SFME) has been demonstrated forextraction of analytes from samples of ultra-small volumes, such as 5μL. This would be suitable for analyzing samples such as blood, whichcan be taken by minimally invasive means such as finger prick. Forsamples available at larger volumes, such as urine or environmentalsamples such as river water, SFME can also be used as shown in FIG. 23.The extraction solvent of small volume, such as 5 or 10 is taken intothe capillary. The solvent can optionally include an internal standard,as shown in FIG. 23. The solvent is either miscible or immiscible withthe sample. In preferred embodiments, the solvent is immiscible with thesample. However, extraction and quantification methods are possible whenthe solvent and sample are miscible with each other. The capillary isthen used to extract the analyte from a liquid sample of relativelylarge volume using slug flow microextraction as shown in FIG. 23. Theextraction solution is then analyzed using nanoESI and a massspectrometer, either sprayed from the capillary or transferred into adifferent hollow body for nanoESI.

The signal intensity of the analyte can be defined as:

$\begin{matrix}{I_{a} = {{k_{a} \cdot C_{a - e}} = {{\frac{k_{a}V_{s}}{{\frac{1}{D_{a}} \cdot V_{s}} + V_{e}} \cdot C_{a - s}^{o}} = {\frac{k_{a}}{\frac{1}{D_{a}} + \frac{V_{e}}{V_{s}}} \cdot C_{a - s}^{o}}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Where ka is the overall response constant, C_(a-e) is the analyteconcentration in the extraction solvent, C_(a-s) ^(o) is the originalanalyte concentration in the sample, V_(s) and V_(e) are the volume ofthe sample and extraction solvent, respectively, and Da is the ratioC_(a-e)/C_(a-s) and C_(a-s) is the analyte concentration in the sampleafter the extraction.

A large volume of the sample may be helpful to improve the sensitivityof the analysis using SFME, as shown in FIGS. 24A-B. Higher intensitiesof the fragment ion peaks from the analytes were obtained with 2 mLsamples, in comparison with 5 or 10 μL samples.

The signal intensity of the analyte can be defined as:

$\begin{matrix}{I_{IS} = {{k_{IS} \cdot C_{{IS} - e}} = {{\frac{k_{IS}V_{e}}{{\frac{1}{D_{IS}} \cdot V_{s}} + V_{e}} \cdot C_{{IS} - e}^{o}} = {\frac{k_{IS}}{{\frac{1}{D_{IS}} \cdot \frac{V_{s}}{V_{e}}} + 1} \cdot C_{a - s}^{o}}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

The I_(a)/I_(IS) ratio can be expressed as:

$\begin{matrix}{\frac{I_{a}}{I_{IS}} = {\frac{k_{a} \cdot C_{a - e}}{k_{IS} \cdot C_{{IS} - e}} = {{\frac{k_{a}}{k_{s}} \cdot \frac{\frac{\mspace{20mu} {1\mspace{14mu} V_{s}}}{D_{IS}V_{e}} + 1}{\frac{1}{D_{a}} + \frac{V_{e}}{V_{s}}} \cdot \frac{C_{a - s}^{o}}{C_{{IS} - e}^{o}}} = {K{\frac{\frac{\mspace{20mu} {1\mspace{14mu} V_{s}}}{D_{IS}V_{e}} + 1}{\frac{1}{D_{a}} + \frac{V_{e}}{V_{s}}} \cdot C_{a - s}^{o}}}}}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

When D_(a) is relatively small and

${V_{s}V_{e}},{{\frac{1}{D_{a}}\frac{V_{e}}{V_{s}}};}$

based on Equation 1, the variations in V_(e) and V_(s) (therebyV_(e)/V_(s)) has minimal impact on I_(a). When

${{\frac{1}{D_{IS}} \cdot \frac{V_{s}}{V_{e}}}{{1,{{viz}.\; D_{{IS}\;}}}}\frac{V_{s}}{V_{e}}},$

the variations in V_(e) and V_(s) (thereby V_(e)/V_(s)) has minimalimpact on the I_(IS) (Equation 2). When these conditions are met, highprecision and accuracy of quantitation are obtained without requiringaccurate or precise measurements of the V_(e) and V_(s). That is,quantification can be performed without knowledge of a volume of thesample and/or solvent.

For a demonstration, amitraz with high log P value (=5) was used as theInternal Standard (IS) for SFME analysis of cotinine (the analyte (A))in urine samples. The V_(e) was randomly selected between 6-9 μL forethyl acetate containing 40 ng/mL amitraz (log P=5) as the extractionsolvent. The V_(s) of urine samples containing 300 ng/mL cotinine (logP=0.07) was selected as 1.0, 1.5, 1.8 and 5 mL. The measured

$\frac{I_{a}}{I_{IS}}$

ratios are shown in FIG. 25. Relatively consistent ratio values wereobtained regardless of the significant changes in the volumes of theextraction solvent and sample. The precision of the measured ratio issignificantly better for a larger volume of 5 mL.

In another demonstration, calibration curve was established with aseries of

$\frac{I_{a}}{I_{IS}}$

measured for urine samples of different volume and containing cotinine(log P=0.07) at different concentration (see FIG. 26 inset). For eachSFME, 10 μL ethyl acetate containing 5 ng/mL verapamil was used as theextraction solvent. Relatively good linearity was obtained, regardlessthe large variations in the sample volume.

It is an important advantage to perform quantitation of an analyte froma sample without requiring the control or knowing the volumes for thesample or the extraction solvent. This makes the on-site and in-fieldanalysis extremely simple.

Example 12 SFME for Analysis of a Target Analyte from Blood

In another example, to extract biomarkers of relatively low polaritiesfrom blood samples, 5 μL sample and 5 μL organic solvent, such as ethylacetate, were injected into a thin capillary. Efficient analyteextraction was achieved with the movement of the liquid plugs. LODs aslow as 0.1 ng/mL have been achieved for analyzing drug compounds inurine and blood samples. For effectively extracting thetenofovir-diphosphate (TFV-DP) of high polarity from the whole bloodlysate in this study, a 3-phase SFME is proposed as shown in FIG. 27A.The analytes are extracted from the polar blood lysate into a polarsolvent, such as methanol:water (50:50), with a nonpolar bridge betweenthem to prevent the salts and cell debris from being extracted. Theextraction solvent can be analyzed using nanoESI directly or ESI throughdirect infusion. In a preliminary test, the 3-phase SFME was performedfor analysis of 10 μL whole blood lysate containing 45 ng/mL TFV-DP. Theextract was analyzed using a TSQ with nanoESI. The MS/MS spectrum wasobtained with characteristic fragment ions m/z 149 and 79 at S/N betterthan 50 (FIG. 27B).

1-20. (canceled)
 21. A method for extracting an analyte from a sample, the method comprising: determining a distribution of an analyte between a sample and a solvent by estimating a partitioning coefficient; selecting the solvent that will extract a target analyte from a sample into the solvent based at least in part on results of the determining step; introducing the solvent into a capillary; introducing the capillary into a vessel comprising the sample such that a portion of the sample is introduced into the capillary; moving the sample and the solvent within the capillary to induce circulation within the sample and the solvent, thereby causing the target analyte to be extracted from the sample and into the solvent, wherein the sample and the solvent do not mix with each other; analyzing the target analyte that has been extracted from the sample; and quantifying the target analyte.
 22. The method according to claim 21, wherein the quantifying step is performed without knowledge of a volume of the sample.
 23. The method according to claim 21, wherein the quantifying step is performed without knowledge of a volume of the solvent.
 24. The method according to claim 21, wherein analyzing comprises: applying a voltage to the solvent comprising the extracted analyte in the capillary so that the analyte is expelled from the capillary, thereby generating ions of the analyte; and analyzing the ions.
 25. The method according to claim 21, wherein analyzing comprises: removing the solvent comprising the extracted analyte from the capillary; and conducting an assay that analyzes the analyte.
 26. The method according to claim 25, wherein the assay comprises: generating ions of the analyte; and analyzing the ions
 27. The method according to claim 21, wherein the solvent comprises an internal standard.
 28. The method according to claim 21, wherein the sample comprises an internal standard.
 29. The method according to claim 21, wherein the solvent is immiscible with the sample.
 30. The method according to claim 21, wherein the solvent is miscible with the sample and the method further comprises introducing a bridging solvent into the capillary that is immiscible with the solvent and the sample in a manner in which the bridging solvent is between the solvent and the sample.
 31. A method for extracting an analyte from a sample, the method comprising: determining a distribution of an analyte between a sample and a solvent by estimating a partitioning coefficient; selecting the solvent that will extract a target analyte from a sample into the solvent based at least in part on results of the determining step; introducing the solvent into a capillary; introducing the capillary into a vessel comprising the sample such that a portion of the sample is introduced into the capillary; moving the sample and the solvent within the capillary to induce circulation within the sample and the solvent, thereby causing the target analyte to be extracted from the sample and into the solvent, wherein the sample and the solvent do not mix with each other.
 32. The method according to claim 31, further comprising analyzing the extracted analyte.
 33. The method according to claim 32, wherein analyzing comprises: applying a voltage to the solvent comprising the extracted analyte in the capillary so that the analyte is expelled from the capillary, thereby generating ions of the analyte; and analyzing the ions.
 34. The method according to claim 32, wherein analyzing comprises: removing the solvent comprising the extracted analyte from the capillary; and conducting an assay that analyzes the analyte.
 35. The method according to claim 34, wherein the assay comprises: generating ions of the analyte; and analyzing the ions
 36. The method according to claim 31, wherein the solvent is introduced to the capillary first.
 37. The method according to claim 31, wherein the sample is introduced to the capillary first.
 38. The method according to claim 31, wherein the solvent comprises an internal standard.
 39. The method according to claim 31, wherein the solvent is immiscible with the sample.
 40. The method according to claim 31, wherein the solvent is miscible with the sample and the method further comprises introducing a bridging solvent into the capillary that is immiscible with the solvent and the sample in a manner in which the bridging solvent is between the solvent and the sample. 